Membrane raft (MR) (formerly lipid raft) in endothelial cell (EC) forms lysosomal-MR signaling platforms or signalosomes to regulate endothelial function and to be involved in endothelial dysfunction, vascular injury and atherogenesis. Over the lasting funding period, we have shown that the MR signaling platforms are associated with translocation of lysosomal acid sphingomyelinase (Asm) and local ceramide production. The present proposal has planned to extend the findings from cell and molecular approaches to animal disease models to address the physiological and pathological relevance of this endothelial MR signaling platform by using genetically-engineered animals. The major focus will be on its triggering role in the activation of nucleotide oligomerization domain-like receptor protein with pryin domain containing 3 (Nlrp3) and consequent endothelial damage and atherosclerotic lesions. The central hypothesis being tested is that Asm-ceramide signaling platforms mediate the activation of Nlrp3 inflammasomes in ECs at the early stage of hypercholesterolemia and thereby produces endothelial injury as a triggering mechanism to result in subsequent atherosclerotic lesions on the carotid arterial wall in concert with local inflammatory responses. To test this hypothesis, three Specific Aims are proposed. Specific aim 1 will determine whether endothelial Nlrp3 inflammasome activation associated with enhanced ceramide production contributes to carotid endothelial dysfunction or injury at the early stage of hypercholesterolemia and late atherosclerotic lesions in the carotid arteries using Asm-/- mice, endothelium-specific Asm transgenic mice (EC-Asmtrg), and their wild type (WT) littermates. Specific Aim 2 will explore the molecular mechanisms by which increased ceramide production activates Nlrp3 inflammasomes with a main focus on the formation of MR redox signalosomes, lysosome dysfunction and kinase suppressor of ras (KSR) as a scaffold in isolated carotid ECs from Asm-/-, EC-Asmtrg and WT mice. In Specific Aim 3, we attempt to determine how ceramide-mediated Nlrp3 inflammasome activation leads to endothelial dysfunction or injury by studying the role of activated caspase-1 and its products in impaired endothelium-dependent vasodilation (EDVD), pyroptosis, altered expression of adhesion and junction proteins, and adaptive endothelial progenitor cells (EPCs) landing or differentiation. To our knowledge, these proposed studies will be the first to investigate the contribution of MR signaling platforms to the activation of Nlrp3 inflammasomes in EC, thereby leading to endothelial dysfunction and consequent atherosclerotic lesion in the arterial wall. The findings of Nlrp3 inflammasome activation to produce both classical inflammatory response and uncanonical vascular injury may shift the paradigm in how we understand the role of inflammation in atherogenesis and cardiovascular diseases.